CN106609266A - Micro-plasminogen variant and micro-plasmin variant obtained from same - Google Patents
Micro-plasminogen variant and micro-plasmin variant obtained from same Download PDFInfo
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- CN106609266A CN106609266A CN201610941060.2A CN201610941060A CN106609266A CN 106609266 A CN106609266 A CN 106609266A CN 201610941060 A CN201610941060 A CN 201610941060A CN 106609266 A CN106609266 A CN 106609266A
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Abstract
The invention relates to a micro-plasminogen variant and a micro-plasmin variant obtained from the same. Specifically, the invention relates to the micro-plasminogen variant and the micro-plasmin variant having one or more amino acid mutations in an enzyme catalytic structural domain, wherein the mutations reduce or prevent micro-plasmin autocatalytic protein hydrolysis. The invention also discloses a pharmaceutical composition using the micro-plasminogen and micro-plasmin variants, and a use and a method thereof.
Description
Technical field
The present invention relates to have the Microplasminogen. and fento of one or more amino acid mutations molten in enzyme catalysiss domain
The variant of enzyme, these mutation reduce or prevent the their catalytic Proteolytic enzyme of MuPlm.
Background technology
Fibrinolytic enzyme, abbreviation fibrinolysin (plasmin) is a kind of by plasminogen (plsminogen) activation
The serine protease of generation.Plasminogen is the important component of mammalian, and human plasminogen is by 791 aminoacid
Composition, molecular weight is about the single chain glycoprotein of 92KDa.Human plasminogen can be activated by plasminogen activator, fibrinolysin
Activator such as urokinase, streptokinase, Sbphylokinase, human histiotype plasminogen activator (tPA), or their variant.Activation
Afterwards, human plasminogen is hydrolyzed at the Arg561-Val562, forms light by a disulfide-bonded heavy chain together and one
Chain.Heavy chain (residue 1-561, about 60kDa) includes 5 Kringle regions (K1～K5), each self-contained about 80 aminoacid
Residue；Light chain (residue 562-791, about 25kDa) is comprising catalyst structure domain.Hydrolysis induction light chain at Arg561-Val562
There is big conformation change, the change cause in light chain by His603, Asp646, the catalytic triads that Ser741 is constituted are opened
Dynamic or activation.
The original various clipped forms of Fibrinolysin (human), people's Miniplasminogen. include the amino of 443～791 of human plasminogen
Sour peptide fragment, is made up of the 5th Kringle region and serine protease domain.Miniplasminogen. can with plasminogen
Identical mode is activated, and forms miniPlm.Human minute plasminogen (microplasminogen), abbreviation people's MuPlm
Original, is by the further clipped form of human plasminogen, N-terminal amino acid peptide fragment (NTP) not including plasminogen, K1～K5
Area, but active region is remained, after being activated, form the small fibrinolysin of people, the fibrinolytic with fibrinolysin.
Fibrinolysin can be as a kind of direct fibrinolytic agent or thrombolytic agent, in the body blood coagulation and fibre
Play a significant role in molten reaction, additionally, fibrinolysin also assists in the activation of metalloproteinases and lipoxygenase class, while
It is also many to body inner cell to migrate related physiology and pathological process correlation, such as cancer, blood vessel hyperplasia, maturation of ovum, embryo
Fetal hair (Crinis Carbonisatus) life, wound healing and cause of disease invade relevant.Additionally, also research prompting fibrinolysin can be used as the auxiliary of vitrectomy
Thing is helped, or extracts the medicament that autologous fibrinolysin (APE) stops excision as pharmacology glass.In sum, fibrinolysin is in body
Highly important effect is played, can be to the disease of therapy-related, with very high medical value.
But human plasminogen or Fibrinolysin (human) also have some to obtain and apply obstacle, the first separation of APE to take time and effort, and
And cost intensive, it is difficult to obtain；Secondly human plasminogen molecular weight is larger, using some host cells such as escherichia coli, suckling
The expression such as zooblast is more difficult, and expression efficiency is than relatively low；Finally as the molecular weight of Fibrinolysin (human) is larger so as to as medicine
Carry out diffusion during treatment organism disease relatively slowly, to affect the treatment.The clipped form of recombinant human plasminogen or Fibrinolysin (human) can be from
It is difficult that the above is solved to a certain extent.Studies have reported that and be used to treat ocular disease using people's MuPlm, or as glass
The ancillary drug of glass Corpectomy, will not be subject to too big to the diffusion of vitreous-body-retina contact surface in the injection position of vitreous body
Hinder (CN 100577202C).
Fibrinolysin is as multiple protein enzyme, it may occur that their catalytic Proteolytic enzyme, i.e. self-dissolving are degraded.Early in nineteen sixty
Early stage in generation, establish fibrinolysin more stable at acidic.Even if but it has been reported that fibrinolysin is stored in pH3.8 conditions
Under, still there is moiety site that self-catalysis degraded occurs, and pH is reduced to a certain extent again, the autocatalysis Degradation meeting of fibrinolysin
Weakening, but have acid hydrolysis factor causes its unstable (W001/36608).Another method is mentioned in patent US5879923
It is addition oligomerised polypeptides complex (oligopeptidic compounds), fibrinolysin can be stablized.Patent WO93/15189 is carried
Fibrinolytic enzyme stability can also be increased to by fibrinolysin Pegylation.But above-mentioned report does not all have to solve fibre from molecular level
The problem of lyase self-dissolving degraded.
Studies have reported that and plasminogen different aminoacids site is mutated, obtain different effects, disclose some
The dependent interaction in site.Dawson et al. describe in patent WO 91/08297 non-degradable plasminogen variant (heavy chain and
Cracking site between light chain is destroyed).It is fine that Dawson et al. Glu replace the Arg (R719E) at 719 reduce Glu-
The affinity (1994, Biochemistry 33,12042-12047) of lyase original variant and streptokinase.Jespers et al. is utilized
Display technique of bacteriophage has screened Microplasminogen. single mutation H569A, R610A, K615A, D660A, Y672A, R712A,
R719A, T782A, R789A, have found 719 sites for the interaction with streptokinase is crucial.Terzyan et al. is reported
Other single mutation (K698M, D740N, S741A) are carried out on the basis of R561A mutation, it is found that these variants can be prevented
The proteolytic activity of plasminogen activation.So as to further prevent activity fibrinolysin formation (2004, Proteins 56,
277-284)。
The mutation of above-mentioned research report does not all reduce the self-dissolving palliating degradation degree of fibrinolysin.And reduce the self-dissolving of fibrinolysin
Palliating degradation degree, preserves for safely and effectively long-term, safely and effectively treats thrombus (thrombolytic therapy)
Or the rear vitreous body in eyes sticks (posterior vitreous detachment) or liquefaction of vitreous (vitreous
Liquefaction) play an important role.
The content of the invention
The present invention relates to the Microplasminogen. of 249 amino acid residues of 543-791 interdigits of human plasminogen sequence, in enzyme
Catalyst structure domain has the Microplasminogen. variant of one or more amino acid mutations, or resultant MuPlm variant,
Or independent MuPlm variant.Described variant is characterized in that in SEQ ID NO:29 hyte propylhomoserins (H) of 2 sequences or/and
The lysine (K) of 103 sports the aminoacid for being not easy to self-dissolving, and on the basis of above-mentioned mutation, 1-4 amino acids can be with one
Or it is multiple disappearance or do not lack.
The sequence of described Microplasminogen. variant is selected from, but is not limited to the sequence in table 1 below
1 Microplasminogen. wild type of table and variant sequence thereof
Another aspect of the present invention is there is provided the MuPlm variant or independent obtained by described Microplasminogen. variant
The MuPlm variant corresponding with Microplasminogen. variant of the present invention.In the present invention, " Microplasminogen. variant is obtained
To MuPlm variant " refer to the fento that Microplasminogen. variant is transformed after plasminogen activator is activated
Lyase variant, the independent MuPlm variant corresponding with Microplasminogen. variant of the present invention are referred to without micro-
Plasminogen variant, and directly obtain otherwise it is corresponding with Microplasminogen. variant provided by the present invention micro-
Fibrinolytic enzyme variants.
Described Microplasminogen. variant, MuPlm variant or their derivant, can reduce the journey of self-catalysis degraded
Degree.Self-catalysis palliating degradation degree detected with chromogenic substrate or biological substrate activity methods, and urged with wild type MuPlm certainly
The degree for changing degraded compares.The self-dissolving degradation constant for being further characterized by the variant of the present invention is wild type MuPlm
At most 50%, and their catalytic constant Kcat is in the range of the 50% to 200% of the Kcat of wild type fibrinolysin.
The invention provides the amino acid mutation of plasminogen catalyst structure domain ad-hoc location, the mutation can reduce or
Fibrinolysin self-dissolving palliating degradation degree, the mutation is prevented to be not limited to Microplasminogen. of the present invention or MuPlm, can
To expand to other plasminogen or fibrinolysin, can be that glutamy plasminogen (Glu-plasminogen) or glutamy are fine
Lyase (Glu-plasmin), lysyl plasminogen (Lys-plasminogen) or lysyl fibrinolysin (Lys-plasmin),
Middle plasminogen (midiplasminogen) or middle fibrinolysin (midiplasmin), Miniplasminogen.
(miniplasminogen) or miniPlm (miniplasmin), Microplasminogen. (microplasminogen) or fento are molten
Enzyme (microplasmin), delta plasminogen (deltaplasminogen) or delta fibrinolysins (delta plasmin).
The invention provides the method for obtaining the Microplasminogen. variant, the method comprising the steps of：
(1) nucleic acid for encoding the Microplasminogen. variant is connected to into expression vector, importing can express the fento
In the former host cell of lyase, wherein described host cell is preferably antibacterial, more preferably escherichia coli, such as BL21 (DE3),
DH5 α etc..Preferably also yeast, more preferably Pichia sp., such as GS115, X33 etc.；Preferably also saccharomyces cerevisiae, such as
INVSc1 etc..
(2) the expression Microplasminogen. of Host Strains described in (1) is made under suitable conditions；
(3) harvest the Microplasminogen. of expression in (2)；
(4) Microplasminogen. in (3) is activated into into MuPlm；
The invention further relates to the nucleotide sequence of encoding plasminogen variant or fibrinolytic enzyme variants, containing these nucleotides sequences
The recombinant vector of row.Convert the part that above-mentioned nucleotide sequence or carrier are also the present invention to host cell.Described host
Cell may be selected from antibacterial or yeast, and the antibacterial is preferably escherichia coli, and the yeast is preferably Pichia sp. or wine brewing
Yeast.
The invention provides the medicine of Microplasminogen. variant, MuPlm variant, variant derivant or any combinations form
Compositions, pharmaceutical composition include conventional adjuvant and additive, or pharmaceutically any acceptable carrier, diluent or steady
Determine agent, and be preferably formulated to pharmaceutical solutionses.
The invention provides reducing the Microplasminogen. variant of self-dissolving palliating degradation degree, MuPlm variant, variant derivant
Or the pharmaceutical composition of any combinations form, for inducing or promoting by the molten of the pathologic fibrin deposition for trying internal
Solution, or the enzymatic debridement of the damaged tissues for reducing recycled fibre proteinogen or for experimenter.
The invention provides reducing the Microplasminogen. variant of self-dissolving palliating degradation degree, MuPlm variant, variant derivant
Or any type of pharmaceutical composition, for preparing the purposes in the medicine for treating eye disorders, wherein eye disorders are selected from such as
Lower disease：Detachment of retina, retinal rupture, vitreous hemorrhage, diabetic vitreous hemorrhage, proliferating diabetic view
Film disease, the non-proliferative diabetic retinopathy degeneration of macula relevant with aging, macula lutea broken hole, the traction of vitreous body macula lutea, Huang
Speckle gauffer, macula lutea exudate, cystoid macular edema, fibrin deposition, the retinal vein occlusion, retinal artery occlusion, regard
Nethike embrane bleed bottom, amblyopia, endophthalmitis, retinopathy of prematurity, glaucoma, retinitis pigmentosa and any of the above group
Close.
Aminoacid three-letter codes and single letter code such as J.biol.chem used by of the invention, in 243, p3558 (1968)
It is described.
Term
" SDS-PAGE electrophoresis " refers to SDS-PAGE, for separating protein.
" HPLC " refers to high performance liquid chromatography, be with liquid as mobile phase, using high pressure transfusion system, will be with different
The mobile phases such as the mixed solvent of the single solvent or different proportion of polarity, buffer pump into the chromatographic column equipped with fixing phase, in post
After interior each composition is separated, detected into detector, so as to realize the analysis to sample.
" Kex2 protease " is a kind of prerequisite knowledge of yeast itself coding, is the serine of a calcium ion dependent form
Protease, its narrow spectrum cutting Lys-Arg, the C-terminal peptide bond of Arg-Arg, Pro-Arg.
Description of the drawings
Fig. 1 is used for the aminoacid sequence of the wild type human Glu- fibrinolysin (1-791) for counting, and the catalytic domain of fibrinolysin
(the 1-230 amino acids that overstriking shows), and 5 Kringle regions have been marked, the wild type Microplasminogen. sequence of the present invention
It is 543-791 amino acids sequences；
The cloning process schematic diagram of Fig. 2 restructuring Microplasminogen .s；
Fig. 3 MuPlms difference duration self-dissolving degraded SDS-PAGE, Microplasminogen. and MuPlm are 27kDa
Left and right, has the impurity of 15kDa or so and 10kDa or so after degraded；
Fig. 4 A Microplasminogen .s HPLC collection of illustrative plates after purification；
Fig. 4 B MuPlms HPLC collection of illustrative plates after purification.
Specific embodiment
It is used for reference to embodiments further describing the present invention, but these embodiments is not intended to limit the scope of the invention.
Embodiment 1:The structure and expression of wild type Microplasminogen. and variant
1. gene chemical synthesis
Wild type human Microplasminogen. nucleotide sequence is synthesized by Nanjing Jin Sirui biotech firms.
The wild type Microplasminogen. nucleotide sequence of synthesis has added an EcoR I at 5 ' ends as shown in SEQ ID NO.1
Restriction enzyme site and Kex2 cleavage sites, and a Not I restriction enzyme site is with the addition of at 3 ' ends.
Italicized item is EcoRI and NotI restriction enzyme sites respectively, and underscore part is Kex2 cleavage sites, and this site is by Kex2
Removing a- factor secretion signal peptides are recognized and cut, ripe albumen is formed.
Corresponding protein sequence is as follows
APSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRP
SSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGET
QGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGC
ARPNKPGVYVRVSRFVTWIEGVMRNN
2. the structure of wild type Microplasminogen. recombinant expression carrier
Expression vector used in the present invention is available from the Pichia sp. secretion expression carrier of Invitrogen companies
pPIC9K(Invitrogen,Cat.K1750-01)。
The wild type Microplasminogen. genetic fragment of synthesis is connected to carrier T test kit (Takara, Cat.D103A)
Carrier T.The above-mentioned carrier T with MuPlm protogene and pPIC9K expression vectors are entered with restriction endonuclease EcoR I and Not I
Double digested, the 37 DEG C of enzyme action 2h of row.Using quick glue reclaim test kit (Wizard SV Gel and PCR Clean-Up
System, Cat#A9281, Lot#0000066824, Progema) gene and carrier segments are reclaimed, connected with T4 ligases
Connect, 16 DEG C of connections are overnight.
Coupled reaction system is as follows：
Take 10 μ l coupled reactions liquid to mix with TOP10 competent cells, it is heat-shock transformed.Culture of the coating with ammonia benzyl resistance
Flat board, picking positive colony extract plasmid and by Nanjing Jin Sirui companies sequence verification, obtain wild type Microplasminogen. MP's
Expression vector.
3. heretofore described amino acid mutation is introduced in wild type MuPlm aminoacid sequence.
In a kind of variant, the histidine of 29 into agedoite, at its corresponding nucleotide sequence 79-81 position
Codon CAC be changed into AAC.Nucleotide sequence after mutation is as follows：
GCTCCATCTTTTGATTGTGGTAAGCCACAAGTTGAACCAAAAAAGTGTCCAGGCAGAGTGGTTGGTGGTTGTGTTGC
TAACCCACATTCTTGGCCTTGGCAAGTTTCTTTGCGTACTAGATTCGGTATGCATTTTTGTGGCGGTACTTTGATTT
CTCCAGAATGGGTTTTGACTGCTGCTCATTGTTTGGAAAAGTCTCCAAGACCATCTTCTTACAAGGTTATTTTGGGT
GCTCATCAGGAGGTCAACCTAGAACCTCATGTTCAAGAAATTGAAGTTTCCAGATTGTTTTTGGAACCAACTAGAAA
GGACATCGCACTTCTTAAATTGTCTTCACCAGCTGTTATTACTGATAAAGTTATTCCAGCTTGTTTGCCATCTCCAA
ACTACGTTGTTGCTGATAGAACTGAATGTTTTATCACTGGTTGGGGTGAAACTCAAGGCACTTTTGGTGCCGGTTTG
TTGAAGGAAGCTCAATTGCCTGTTATAGAAAACAAAGTTTGTAACAGATACGAATTTTTGAACGGTAGAGTTCAATC
TACTGAATTGTGTGCTGGTCATTTGGCTGGTGGTACTGATTCTTGTCAAGGTGATAGTGGAGGTCCATTGGTCTGTT
TTGAAAAAGATAAGTACATTTTGCAAGGTGTTACTTCTTGGGGTTTGGGTTGTGCTAGACCAAATAAACCAGGTGTT
TACGTTAGAGTTTCTAGATTTGTCACTTGGATTGAGGGTGTTATGAGAAACAACTAA SEQ NO ID.3
Its corresponding aminoacid sequence is as follows, i.e. MP1 variant sequence thereofs：
APSFDCGKPQVEPKKCPGRVVGGCVAHPNSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILG
AHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGL
LKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGV
YVRVSRFVTWIEGVMRNN SEQ NO ID.4
In another kind of variant, the lysine of 103 is replaced as into alanine, by its corresponding nucleotide sequence 314-
Codon AAG at 316 is changed into GCT.Nucleotide sequence after mutation is as follows：
GCTCCATCTTTTGATTGTGGTAAGCCACAAGTTGAACCAAAAAAGTGTCCAGGCAGAGTGGTTGGTGGTTGTGTTGC
TCACCCACATTCTTGGCCTTGGCAAGTTTCTTTGCGTACTAGATTCGGTATGCATTTTTGTGGCGGTACTTTGATTT
CTCCAGAATGGGTTTTGACTGCTGCTCATTGTTTGGAAAAGTCTCCAAGACCATCTTCTTACAAGGTTATTTTGGGT
GCTCATCAGGAGGTCAACCTAGAACCTCATGTTCAAGAAATTGAAGTTTCCAGATTGTTTTTGGAACCAACTAGAAA GGACATCGCACTTCTTAAATTGTCTTCACCAGCTGTTATTACTGATAAAGTTATTCCAGCTTGTTTGCCATCTCCAA
ACTACGTTGTTGCTGATAGAACTGAATGTTTTATCACTGGTTGGGGTGAAACTCAAGGCACTTTTGGTGCCGGTTTG
TTGAAGGAAGCTCAATTGCCTGTTATAGAAAACAAAGTTTGTAACAGATACGAATTTTTGAACGGTAGAGTTCAATC
TACTGAATTGTGTGCTGGTCATTTGGCTGGTGGTACTGATTCTTGTCAAGGTGATAGTGGAGGTCCATTGGTCTGTT
TTGAAAAAGATAAGTACATTTTGCAAGGTGTTACTTCTTGGGGTTTGGGTTGTGCTAGACCAAATAAACCAGGTGTT
TACGTTAGAGTTTCTAGATTTGTCACTTGGATTGAGGGTGTTATGAGAAACAACTAA SEQ NO ID.5
Its corresponding aminoacid sequence is as follows, i.e. MP2 variant sequence thereofs：
APSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILG
AHQEVNLEPHVQEIEVSRLFLEPTRADIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGL
LKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGV
YVRVSRFVTWIEGVMRNN SEQ NO ID.6
In addition, by the histidine of 29 into agedoite, the lysine of 103 is replaced as alanine；Corresponded to
Nucleotide sequence 79-81 positions at codon CAC be changed into AAC；Codon at its corresponding nucleotide sequence 314-316 position
AAG is changed into GCT.Nucleotide sequence after mutation is as follows：
GCTCCATCTTTTGATTGTGGTAAGCCACAAGTTGAACCAAAAAAGTGTCCAGGCAGAGTGGTTGGTGGTTGTGTTGC
TAACCCACATTCTTGGCCTTGGCAAGTTTCTTTGCGTACTAGATTCGGTATGCATTTTTGTGGCGGTACTTTGATTT
CTCCAGAATGGGTTTTGACTGCTGCTCATTGTTTGGAAAAGTCTCCAAGACCATCTTCTTACAAGGTTATTTTGGGT
GCTCATCAGGAGGTCAACCTAGAACCTCATGTTCAAGAAATTGAAGTTTCCAGATTGTTTTTGGAACCAACTAGAAA GGACATCGCACTTCTTAAATTGTCTTCACCAGCTGTTATTACTGATAAAGTTATTCCAGCTTGTTTGCCATCTCCAA
ACTACGTTGTTGCTGATAGAACTGAATGTTTTATCACTGGTTGGGGTGAAACTCAAGGCACTTTTGGTGCCGGTTTG
TTGAAGGAAGCTCAATTGCCTGTTATAGAAAACAAAGTTTGTAACAGATACGAATTTTTGAACGGTAGAGTTCAATC
TACTGAATTGTGTGCTGGTCATTTGGCTGGTGGTACTGATTCTTGTCAAGGTGATAGTGGAGGTCCATTGGTCTGTT
TTGAAAAAGATAAGTACATTTTGCAAGGTGTTACTTCTTGGGGTTTGGGTTGTGCTAGACCAAATAAACCAGGTGTT
TACGTTAGAGTTTCTAGATTTGTCACTTGGATTGAGGGTGTTATGAGAAACAACTAA SEQ NO ID.7
Its corresponding aminoacid sequence is as follows, i.e. MP3 variant sequence thereofs：
APSFDCGKPQVEPKKCPGRVVGGCVAHPNSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILG
AHQEVNLEPHVQEIEVSRLFLEPTRADIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGL
LKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGV
YVRVSRFVTWIEGVMRNN SEQ NO ID.8
On the basis of above-mentioned mutant, the aminoacid of 1 or first 4 is lacked, i.e., the Histidine mutagenesis of 29
Lysine mutation for agedoite or/and 103 is alanine, while the aminoacid of disappearance 1 or first 4.At 29
Histidine mutagenesis lack the 1st alanine while being agedoite, obtain Microplasminogen. variant MP4, its aminoacid sequence
It is as follows：
PSFDCGKPQVEPKKCPGRVVGGCVAHPNSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGA
HQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLL
KEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVY
VRVSRFVTWIEGVMRNN SEQ NO ID.9
While the Histidine mutagenesis of 29 are agedoite, front 4 amino acids of disappearance, obtain Microplasminogen. variant
MP5, its aminoacid sequence are as follows：
DCGKPQVEPKKCPGRVVGGCVAHPNSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQE
VNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEA
QLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRV
SRFVTWIEGVMRNN SEQ NO ID.10
The 1st alanine is lacked while the lysine mutations of 103 are alanine, Microplasminogen. variant is obtained
MP6, its aminoacid sequence are as follows：
PSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGA
HQEVNLEPHVQEIEVSRLFLEPTRADIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLL
KEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVY
VRVSRFVTWIEGVMRNN SEQ NO ID.11
While the lysine mutations of 103 are alanine, front 4 amino acids of disappearance, obtain Microplasminogen. variant
MP7, its aminoacid sequence are as follows：
DCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQE
VNLEPHVQEIEVSRLFLEPTRADIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEA
QLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRV
SRFVTWIEGVMRNN SEQ NO ID.12
29 H sport N, 103 K and sport A, and lack the 1st alanine, obtain Microplasminogen. variant MP8,
Its aminoacid sequence is as follows：
PSFDCGKPQVEPKKCPGRVVGGCVAHPNSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGA
HQEVNLEPHVQEIEVSRLFLEPTRADIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLL
KEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVY
VRVSRFVTWIEGVMRNN SEQ ID NO.13
29 H sport N, 103 K and sport A, and lack front 4 amino acids, obtain Microplasminogen. variant MP9,
Its aminoacid sequence is as follows：
DCGKPQVEPKKCPGRVVGGCVAHPNSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQE
VNLEPHVQEIEVSRLFLEPTRADIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEA
QLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRV
SRFVTWIEGVMRNN SEQ ID NO.14
On the basis of wild type Microplasminogen. expression vector, introduce single mutation or double mutation to obtain with KOD PCR methods
The expression vector of MP1-MP3 Microplasminogen. variants, KOD PCR methods are using KOD test kits (TOYOBO, Cat.KOD-201)
Come what is carried out, primer is as shown in table 2.
2 Microplasminogen. different loci of table is mutated the primer sequence
With SEQ ID NO.15 and SEQ ID NO.16 as primer, KOD PCR are carried out by template of the expression vector of MP, obtained
MP1 expression vectors are obtained, KOD PCR systems are as follows：
Amplification program：
94 DEG C of 2min,
94 DEG C of 30s, 55 DEG C of 30s, 68 DEG C of 10min, expand 25 circulations
68 DEG C of incubation 10min
Terminate PCR amplification programs
After PCR reactions terminate, 2 μ L restriction endonuclease Dpn I are added in PCR reactant liquors (50 μ L of total amount), is mixed, 37 DEG C of reactions
1h.Take 10 μ L reactant liquors to mix with TOP10 competent cells, it is heat-shock transformed, Amp culture plates are coated with, picking positive colony is taken out
Upgrading grain, send Nanjing Jin Sirui companies to carry out sequence verification, obtains the expression vector of MP1.
With SEQ ID NO.17 and SEQ ID NO.18 as primer, KOD PCR are carried out by template of the expression vector of MP, obtained
MP2 expression vectors are obtained, condition and method are consistent with above-mentioned MP1 expression vectors acquisition methods.
With SEQ ID NO.15 and SEQ ID NO.16 as primer, KOD PCR are carried out by template of the expression vector of MP2,
MP3 expression vectors are obtained, condition and method are consistent with above-mentioned MP1 expression vectors acquisition methods.
The disappearance of wild type Microplasminogen. first or front 4 amino acids is introduced with the method for PCR, MP4-MP9 is obtained
Microplasminogen. variant, primer are as shown in table 3.
3 Microplasminogen. different loci aminoacid deletion the primer sequence of table
With corresponding sequence as primer, performing PCR is entered as masterplate with the expression vector of MP,
PCR conditions：
94 DEG C of 3min,
94 DEG C of 30s, 59 DEG C of 50s, 72 DEG C of 30s, expand 30 circulations
72 DEG C, 5min
Terminate PCR amplification programs
PCR fragment is reclaimed using glue reclaim test kit, T loads is connected to carrier T test kit (Takara, Cat.D103A)
Body.The above-mentioned carrier T with Microplasminogen. variant gene and pPIC9K expression vectors are entered with restriction endonuclease EcoR I and Not I
Row is double digested.Gene and carrier segments are reclaimed using quick glue reclaim test kit, is then connected using quick ligase, turned
Change to TOP10 competent cells, be coated with Amp resistance culture flat boards, picking single bacterium colony, extracting plasmid order-checking analysis.Concrete steps
Such as the structure of wild type Microplasminogen. expression vector.
4. wild type Microplasminogen. and variant is recombinant expressed
Above-mentioned recombinant expression carrier is transformed into into the GS115 Pichi strains from the purchase of invitrogen companies
(invitrogen, Cat.K1750-01), structure obtain the recombinant bacterium of Microplasminogen. variant, and recombinant bacterium is coated on containing G418
The YPD flat boards of resistance are screened.Conversion and screening technique step are operated referring to the Pichia anomala expression of invitrogen companies
Handbook (invitrogen, Cat.K1750-01).
In 50mL BMGY culture medium, 30 DEG C, 250rpm cultivates 20-24h to single bacterium colony after picking G418 screenings.Detection
OD600Value, should be between 2-6.Cell, Ran Houyong are collected with centrifuge (Beckman Coulter) 3000rpm centrifugation 5min
BMMY culture medium is resuspended to OD600For 1, absolute methanol (analysis is pure) final concentration of 0.5% in culture medium, 30 DEG C, 250rpm cultures
72h, adds the absolute methanol of 1/200 cumulative volume per 12h, and 1mL, 12000rpm centrifugation 5min are sampled per 24h, supernatant is collected.
SDS-PAGE (invitrogen, Cat.No.456-1083) electrophoresis detection destination protein is expressed.
Embodiment 2:The 5L fermentation tank scale fermentations of Microplasminogen.
Following 4 step of 5L scale fermentations point is carried out.
1. take 2ml glycerol stocks be seeded to equipped with 200ml YPG culture medium (yeast powder 10g/L, soy peptone 20g/L, it is sweet
Oily 20g/L) 1L shaking flasks in, the 250rpm shaken cultivation about 22h at 30 DEG C is cultivated to strain OD600For 12 or so.
2. cultured seed liquor is seeded in 5L fermentation tanks and is fermented, using 2.5L BSM culture medium (its component
For：85% phosphoric acid 26.7ml/L, CaSO4·2H2O 1.2g/L, K2SO418.2g/L, MgSO47.3g/L, KOH 4.1g/L is sweet
Oily 40.0g/L, PTM1 4.4ml/L, 25% ammonia adjust pH5.8.PTM1:CuSO4·5H2O, 6.0g/L；KI, 0.08g/L；
MnSO4·H2O, 3.0g/L；H3BO3, 0.02g/L；MoNa2O4·2H2O, 0.2g/L；CoCl2, 0.5g/L；ZnCl2, 20.0g/L；
FeSO4·7H2O, 65.0g/L；Biotin, 0.2g/L；H2SO4, 5.0ml/L), cultivation temperature is set as 30 DEG C, and ventilation keeps
6L/min, adjustment speed of agitator make DO (dissolved oxygen concentration)>20%, pH is adjusted with 25% ammonia, maintain pH 5.8.
3. when cultivating to glycerol depletion, DO is raised rapidly, and (50% glycerol contains now to start stream glycerol adding supplemented medium
1.2%PTM1), adjusting speed of agitator makes DO (dissolved oxygen concentration)>20%, pH is adjusted with 25% ammonia, maintain pH 5.8.Training
Support to strain OD600When reaching 150 or so, stop glycerol feed supplement.
4. after glycerol feed supplement terminates, disposable addition absolute methanol 46mL, make methanol concentration in fermentation liquid reach 0.2%～
0.3%.After strain adapts to methanol environment, cultivation temperature is set to into 25 DEG C, culture pH is set to 5.8, starts methanol induction,
Methanol feeding culture medium (absolute methanol contains 1.2%PTM1) is filled into, according to dissolved oxygen feedback adjustment methanol feeding speed, DO is maintained
(dissolved oxygen concentration)>20%, induce 48h, fermentation ends.
Embodiment 3:The purification of Microplasminogen. and MuPlm
Fermentation culture again use respectively after a step doughnut clarification step cation-exchange chromatography and step the moon from
The purifying procedure of two steps of sub- displacement chromatography obtains Microplasminogen..MuPlm after activation through hydrophobic chromatography, concentration,
Displacement three step of buffer obtains the MuPlm (CN100577202C) of purification.
Embodiment 4：The activation of Microplasminogen. and its variant
Plasminogen is diluted to into 0.5g/L-1.0g/L (18 μM -36 μM), plasminogen with the mol ratio of Sbphylokinase is
(50-200):1,25 DEG C of reaction 30min, adds pre-cooling ammonium sulfate and tranexamic acid, makes the final concentration of 1M of ammonium sulfate, tranexamic acid
Final concentration of 0.1M, in order to stabilize MuPlm, is stored on ice, timely purification.By determine Microplasminogen. initial concentration and
The concentration of activated MuPlm after activation, final Microplasminogen. and variant activity ratio are all more than 90%.
Embodiment 5：Determine the self-dissolving degradation constant of wild type MuPlm and variant
Self-dissolving constant K is a secondary rate constant, is determined using following method：
1. the Microplasminogen. variant of purification is activated using recombinant glucokinase (ENZ-288, Prospec company), is allowed to turn
The MuPlm of chemical conversion activity.Microplasminogen. is diluted to into 0.2g/L-0.5g/L (7 μM -18 μM), plasminogen and Sbphylokinase
Mol ratio be 50:1 (mass ratio is 88:1), 37 DEG C of reaction 10min, now to have activated into fento completely molten for Microplasminogen.
Enzyme, and there is few self-dissolving degradation reaction.At regular intervals afterwards (0min, 5min, 10min, 20min, 30min, 1h,
2h, 3h, 6h) 100 μ L samples are taken out, 200 μ l pre-cooling 5mM citric acid terminating reactions are added, is placed in and is preserved on ice；
2. the peak area of all time point samples is detected with HPLC, the molar concentration of remaining MuPlm is calculated (with wild
The Microplasminogen. purification product of type set up the standard curve of concentration correspondence peak area).Or by the MuPlm diluted sample for being taken
(MuPlm final concentration is controlled in 10nM-30nM) adds 0.5mM chromogenic substrate Glu-Phe-Lys-pNA in 96 orifice plates afterwards
In (S2403, Chromogenix, Milan), 96 orifice plates are positioned in the microplate reader of 37 DEG C of preheating and are reacted, select 405nm ripples
It is long, a secondary data is read per 30s, 10min is read.By decline degree of the MuPlm to the hydrolysing activity of S2403, calculate
The molar concentration of remaining MuPlm.
3. the degradation curve of the concentration (mol/L) with the time (s) of remaining MuPlm is drawn, and using nonlinear regression point
Analysis, data are brought in equation, obtain self-dissolving degradation constant k (M-1s-1)。
Formula 1：
In formula, k is that (unit is M to self-dissolving degradation constant-1s-1), t is enzyme reaction time (unit is s), CMPIt is any
The concentration (unit is mol/L) of MuPlm, C at given time pointMP0It is the concentration of MuPlm at t=0.
Experimental result, the self-dissolving degradation constant k of MuPlm mutant of the present invention is wild type human MuPlm
10%-50%, illustrate the present invention Microplasminogen. and fibrinolytic enzyme mutant can reduce fibrinolysin self-catalysis degraded journey
Degree.The self-dissolving constant k value of MuPlm and its variant is shown in Table 4.
The self-dissolving degradation constant k value of 4 MuPlm of table and its variant
| Title | Orresponding amino acid serial number | Mutated site | Self-dissolving degradation constant k (M-1s-1) |
| MP | SEQ ID NO:2 | Wild type | 120 |
| MP1 | SEQ ID NO:4 | H29N | 56 |
| MP2 | SEQ ID NO:6 | K103A | 35 |
| MP3 | SEQ ID NO:8 | H29NK103A | 11 |
| MP4 | SEQ ID NO:9 | H29N, lacks 1A | 42 |
| MP5 | SEQ ID NO:10 | H29N, lacks 1A2P3S4F | 39 |
| MP6 | SEQ ID NO:11 | K103A, lacks 1A | 37 |
| MP7 | SEQ ID NO:12 | K103A, lacks 1A2P3S4F | 30 |
| MP8 | SEQ ID NO:13 | H29NK103A, lacks 1A | 12 |
| MP9 | SEQ ID NO:14 | H29NK103A, lacks 1A2P3S4F | 10 |
Embodiment 6：Determine the proteolytic activity of MuPlm variant
Principle：By detecting MuPlm to chromogenic substrate S-2403, hydrolysing activity, determine Km, Kcat.S-2403
After hydrolysis, product pNA (paranitroanilinum) is generated, causes the absorbance at 405nm to increase, using microplate reader (Bio-Tek)
The change of the absorbance at 405nm is detected, further according to the standard curve of the content of absorbance correspondence pNA, primary response speed is calculated
Rate, so as to obtain the enzyme kineticss parameter of correlation.
To obtain the MuPlm of activity, activated using Sbphylokinase, Sbphylokinase is 1 with plasminogen mol ratio:50, control
At 0.2g/L-0.5g/L (7 μM -18 μM), buffer solution system is 25mM disodium hydrogen phosphates to MuPlm original content, 50mM NaCl,
PH7.0,37 DEG C of incubation 10min, adds the 5mM citric acids of the pre-cooling of 2 times of volumes, pH is reduced to about 3, and is stored in 4 DEG C,
Terminating reaction simultaneously makes MuPlm stable.Arrange substrate S2403 variable concentrations (2mM, 1mM, 0.5mM, 0.25mM,
0.125mM, 0.0625mM, 0.03125mM, 0.015625mM), the substrate of each concentration repeats 3 holes.Microplate reader is preheated to 37
DEG C, 405nm wavelength is selected, a secondary data is read per 30s, 10min is read.The S2403 of each concentration has 3 multiple holes, absorbance
Average, deduct background light absorption value, according to the standard curve of the content of absorbance correspondence pNA, calculate initial hydrolysis rate.Again
By below equation analytical data, obtain MuPlm variant to the enzyme kineticss parameter of chromogenic substrate S-2403 (Km,
Kcat), wherein CMPIt is the concentration (mol/L) of the active MuPlm detected by HPLC
Formula 2：
Formula 3：
In formula, v is initial reaction rate (unit is a μm ol/s), and Km is Michaelis constant (unit be μM), and Kcat is
(unit is s to catalysic coefficient-1), Vmax be enzymatic reaction maximum rate, CMPIt is the dense of MuPlm at any given time point
Degree, CMP0It is the concentration of MuPlm at t=0.
As a result show, Michaelis constant Km of MuPlm variant of the present invention is the 96%- of wild type MuPlm
120%, illustrate that MuPlm variant of the present invention is suitable with wild type to substrate binding ability.Fento of the present invention
The catalysic coefficient Kcat of lyase variant is the 70%-140% of wild type MuPlm, illustrates MuPlm of the present invention
The catalytic efficiency of variant is not significantly reduced with wild type MuPlm, or even is improved.MuPlm and its variant
Enzyme kineticss parameter (Km, Kcat) see the table below
The Km Kcat values of 5 MuPlm of table and its variant
Sequence table
<110>Hengrui Medicine Co., Ltd., Jiangsu Prov.
<120>A kind of Microplasminogen. variant and the MuPlm variant obtained by which
<130> 360268CG-350169
<160> 22
<170> PatentIn version 3.3
<210> 1
<211> 770
<212> DNA
<213>Artificial sequence
<400> 1
gaattcaaga gagctccatc ttttgattgt ggtaagccac aagttgaacc aaaaaagtgt 60
ccaggcagag tggttggtgg ttgtgttgct cacccacatt cttggccttg gcaagtttct 120
ttgcgtacta gattcggtat gcatttttgt ggcggtactt tgatttctcc agaatgggtt 180
ttgactgctg ctcattgttt ggaaaagtct ccaagaccat cttcttacaa ggttattttg 240
ggtgctcatc aggaggtcaa cctagaacct catgttcaag aaattgaagt ttccagattg 300
tttttggaac caactagaaa ggacatcgca cttcttaaat tgtcttcacc agctgttatt 360
actgataaag ttattccagc ttgtttgcca tctccaaact acgttgttgc tgatagaact 420
gaatgtttta tcactggttg gggtgaaact caaggcactt ttggtgccgg tttgttgaag 480
gaagctcaat tgcctgttat agaaaacaaa gtttgtaaca gatacgaatt tttgaacggt 540
agagttcaat ctactgaatt gtgtgctggt catttggctg gtggtactga ttcttgtcaa 600
ggtgatagtg gaggtccatt ggtctgtttt gaaaaagata agtacatttt gcaaggtgtt 660
acttcttggg gtttgggttg tgctagacca aataaaccag gtgtttacgt tagagtttct 720
agatttgtca cttggattga gggtgttatg agaaacaact aagcggccgc 770
<210> 2
<211> 249
<212> PRT
<213>Artificial sequence
<400> 2
Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys
1 5 10 15
Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro
20 25 30
Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly
35 40 45
Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu
50 55 60
Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln
65 70 75 80
Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu
85 90 95
Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser
100 105 110
Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro
115 120 125
Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly
130 135 140
Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu
145 150 155 160
Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly
165 170 175
Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr
180 185 190
Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys
195 200 205
Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala
210 215 220
Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr
225 230 235 240
Trp Ile Glu Gly Val Met Arg Asn Asn
245
<210> 3
<211> 750
<212> DNA
<213>Artificial sequence
<400> 3
gctccatctt ttgattgtgg taagccacaa gttgaaccaa aaaagtgtcc aggcagagtg 60
gttggtggtt gtgttgctaa cccacattct tggccttggc aagtttcttt gcgtactaga 120
ttcggtatgc atttttgtgg cggtactttg atttctccag aatgggtttt gactgctgct 180
cattgtttgg aaaagtctcc aagaccatct tcttacaagg ttattttggg tgctcatcag 240
gaggtcaacc tagaacctca tgttcaagaa attgaagttt ccagattgtt tttggaacca 300
actagaaagg acatcgcact tcttaaattg tcttcaccag ctgttattac tgataaagtt 360
attccagctt gtttgccatc tccaaactac gttgttgctg atagaactga atgttttatc 420
actggttggg gtgaaactca aggcactttt ggtgccggtt tgttgaagga agctcaattg 480
cctgttatag aaaacaaagt ttgtaacaga tacgaatttt tgaacggtag agttcaatct 540
actgaattgt gtgctggtca tttggctggt ggtactgatt cttgtcaagg tgatagtgga 600
ggtccattgg tctgttttga aaaagataag tacattttgc aaggtgttac ttcttggggt 660
ttgggttgtg ctagaccaaa taaaccaggt gtttacgtta gagtttctag atttgtcact 720
tggattgagg gtgttatgag aaacaactaa 750
<210> 4
<211> 249
<212> PRT
<213>Artificial sequence
<400> 4
Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys
1 5 10 15
Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro Asn Ser Trp Pro
20 25 30
Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly
35 40 45
Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu
50 55 60
Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln
65 70 75 80
Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu
85 90 95
Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser
100 105 110
Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro
115 120 125
Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly
130 135 140
Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu
145 150 155 160
Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly
165 170 175
Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr
180 185 190
Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys
195 200 205
Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala
210 215 220
Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr
225 230 235 240
Trp Ile Glu Gly Val Met Arg Asn Asn
245
<210> 5
<211> 750
<212> DNA
<213>Artificial sequence
<400> 5
gctccatctt ttgattgtgg taagccacaa gttgaaccaa aaaagtgtcc aggcagagtg 60
gttggtggtt gtgttgctca cccacattct tggccttggc aagtttcttt gcgtactaga 120
ttcggtatgc atttttgtgg cggtactttg atttctccag aatgggtttt gactgctgct 180
cattgtttgg aaaagtctcc aagaccatct tcttacaagg ttattttggg tgctcatcag 240
gaggtcaacc tagaacctca tgttcaagaa attgaagttt ccagattgtt tttggaacca 300
actagaaagg acatcgcact tcttaaattg tcttcaccag ctgttattac tgataaagtt 360
attccagctt gtttgccatc tccaaactac gttgttgctg atagaactga atgttttatc 420
actggttggg gtgaaactca aggcactttt ggtgccggtt tgttgaagga agctcaattg 480
cctgttatag aaaacaaagt ttgtaacaga tacgaatttt tgaacggtag agttcaatct 540
actgaattgt gtgctggtca tttggctggt ggtactgatt cttgtcaagg tgatagtgga 600
ggtccattgg tctgttttga aaaagataag tacattttgc aaggtgttac ttcttggggt 660
ttgggttgtg ctagaccaaa taaaccaggt gtttacgtta gagtttctag atttgtcact 720
tggattgagg gtgttatgag aaacaactaa 750
<210> 6
<211> 249
<212> PRT
<213>Artificial sequence
<400> 6
Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys
1 5 10 15
Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro
20 25 30
Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly
35 40 45
Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu
50 55 60
Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln
65 70 75 80
Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu
85 90 95
Phe Leu Glu Pro Thr Arg Ala Asp Ile Ala Leu Leu Lys Leu Ser Ser
100 105 110
Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro
115 120 125
Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly
130 135 140
Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu
145 150 155 160
Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly
165 170 175
Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr
180 185 190
Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys
195 200 205
Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala
210 215 220
Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr
225 230 235 240
Trp Ile Glu Gly Val Met Arg Asn Asn
245
<210> 7
<211> 750
<212> DNA
<213>Artificial sequence
<400> 7
gctccatctt ttgattgtgg taagccacaa gttgaaccaa aaaagtgtcc aggcagagtg 60
gttggtggtt gtgttgctaa cccacattct tggccttggc aagtttcttt gcgtactaga 120
ttcggtatgc atttttgtgg cggtactttg atttctccag aatgggtttt gactgctgct 180
cattgtttgg aaaagtctcc aagaccatct tcttacaagg ttattttggg tgctcatcag 240
gaggtcaacc tagaacctca tgttcaagaa attgaagttt ccagattgtt tttggaacca 300
actagaaagg acatcgcact tcttaaattg tcttcaccag ctgttattac tgataaagtt 360
attccagctt gtttgccatc tccaaactac gttgttgctg atagaactga atgttttatc 420
actggttggg gtgaaactca aggcactttt ggtgccggtt tgttgaagga agctcaattg 480
cctgttatag aaaacaaagt ttgtaacaga tacgaatttt tgaacggtag agttcaatct 540
actgaattgt gtgctggtca tttggctggt ggtactgatt cttgtcaagg tgatagtgga 600
ggtccattgg tctgttttga aaaagataag tacattttgc aaggtgttac ttcttggggt 660
ttgggttgtg ctagaccaaa taaaccaggt gtttacgtta gagtttctag atttgtcact 720
tggattgagg gtgttatgag aaacaactaa 750
<210> 8
<211> 249
<212> PRT
<213>Artificial sequence
<400> 8
Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys
1 5 10 15
Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro Asn Ser Trp Pro
20 25 30
Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly
35 40 45
Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu
50 55 60
Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln
65 70 75 80
Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu
85 90 95
Phe Leu Glu Pro Thr Arg Ala Asp Ile Ala Leu Leu Lys Leu Ser Ser
100 105 110
Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro
115 120 125
Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly
130 135 140
Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu
145 150 155 160
Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly
165 170 175
Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr
180 185 190
Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys
195 200 205
Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala
210 215 220
Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr
225 230 235 240
Trp Ile Glu Gly Val Met Arg Asn Asn
245
<210> 9
<211> 248
<212> PRT
<213>Artificial sequence
<400> 9
Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro
1 5 10 15
Gly Arg Val Val Gly Gly Cys Val Ala His Pro Asn Ser Trp Pro Trp
20 25 30
Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr
35 40 45
Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys
50 55 60
Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu
65 70 75 80
Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe
85 90 95
Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro
100 105 110
Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn
115 120 125
Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu
130 135 140
Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro
145 150 155 160
Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg
165 170 175
Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp
180 185 190
Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp
195 200 205
Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg
210 215 220
Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp
225 230 235 240
Ile Glu Gly Val Met Arg Asn Asn
245
<210> 10
<211> 245
<212> PRT
<213>Artificial sequence
<400> 10
Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val
1 5 10 15
Val Gly Gly Cys Val Ala His Pro Asn Ser Trp Pro Trp Gln Val Ser
20 25 30
Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser
35 40 45
Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg
50 55 60
Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu
65 70 75 80
Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro
85 90 95
Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile
100 105 110
Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val
115 120 125
Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly
130 135 140
Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu
145 150 155 160
Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser
165 170 175
Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln
180 185 190
Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile
195 200 205
Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys
210 215 220
Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly
225 230 235 240
Val Met Arg Asn Asn
245
<210> 11
<211> 248
<212> PRT
<213>Artificial sequence
<400> 11
Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro
1 5 10 15
Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp
20 25 30
Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr
35 40 45
Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys
50 55 60
Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu
65 70 75 80
Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe
85 90 95
Leu Glu Pro Thr Arg Ala Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro
100 105 110
Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn
115 120 125
Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu
130 135 140
Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro
145 150 155 160
Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg
165 170 175
Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp
180 185 190
Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp
195 200 205
Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg
210 215 220
Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp
225 230 235 240
Ile Glu Gly Val Met Arg Asn Asn
245
<210> 12
<211> 245
<212> PRT
<213>Artificial sequence
<400> 12
Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val
1 5 10 15
Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser
20 25 30
Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser
35 40 45
Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg
50 55 60
Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu
65 70 75 80
Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro
85 90 95
Thr Arg Ala Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile
100 105 110
Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val
115 120 125
Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly
130 135 140
Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu
145 150 155 160
Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser
165 170 175
Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln
180 185 190
Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile
195 200 205
Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys
210 215 220
Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly
225 230 235 240
Val Met Arg Asn Asn
245
<210> 13
<211> 248
<212> PRT
<213>Artificial sequence
<400> 13
Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro
1 5 10 15
Gly Arg Val Val Gly Gly Cys Val Ala His Pro Asn Ser Trp Pro Trp
20 25 30
Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr
35 40 45
Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys
50 55 60
Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu
65 70 75 80
Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe
85 90 95
Leu Glu Pro Thr Arg Ala Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro
100 105 110
Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn
115 120 125
Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu
130 135 140
Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro
145 150 155 160
Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg
165 170 175
Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp
180 185 190
Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp
195 200 205
Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg
210 215 220
Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp
225 230 235 240
Ile Glu Gly Val Met Arg Asn Asn
245
<210> 14
<211> 245
<212> PRT
<213>Artificial sequence
<400> 14
Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val
1 5 10 15
Val Gly Gly Cys Val Ala His Pro Asn Ser Trp Pro Trp Gln Val Ser
20 25 30
Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser
35 40 45
Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg
50 55 60
Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu
65 70 75 80
Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro
85 90 95
Thr Arg Ala Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile
100 105 110
Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val
115 120 125
Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly
130 135 140
Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu
145 150 155 160
Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser
165 170 175
Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln
180 185 190
Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile
195 200 205
Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys
210 215 220
Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly
225 230 235 240
Val Met Arg Asn Asn
245
<210> 15
<211> 36
<212> DNA
<213>Artificial sequence
<400> 15
gagtggttgg tggttgtgtt gctaacccac attctt 36
<210> 16
<211> 36
<212> DNA
<213>Artificial sequence
<400> 16
aagaatgtgg gttagcaaca caaccaccaa ccactc 36
<210> 17
<211> 55
<212> DNA
<213>Artificial sequence
<400> 17
ccagattgtt tttggaacca actagagctg acatcgcact tcttaaattg tcttc 55
<210> 18
<211> 55
<212> DNA
<213>Artificial sequence
<400> 18
gaagacaatt taagaagtgc gatgtcagct ctagttggtt ccaaaaacaa tctgg 55
<210> 19
<211> 37
<212> DNA
<213>Artificial sequence
<400> 19
ccggaattca agagaccatc ttttgattgt ggtaagc 37
<210> 20
<211> 31
<212> DNA
<213>Artificial sequence
<400> 20
ggcaaatggc attctgacat cctattgatt a 31
<210> 21
<211> 34
<212> DNA
<213>Artificial sequence
<400> 21
ccggaattca agagagattg tggtaagcca caag 34
<210> 22
<211> 31
<212> DNA
<213>Artificial sequence
<400> 22
ggcaaatggc attctgacat cctattgatt a 31
Claims (14)
1. a kind of Microplasminogen. variant, wherein histidine (H) in Microplasminogen. 29, the lysine (K) of 103 can be with
The aminoacid for being not easy to self-dissolving is individually or simultaneously sported, and 1-4 amino acids there can be one or more disappearances or not lack
Lose, described Microplasminogen. sequence is as follows：
APSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYK
VILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTF
GAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPN
KPGVYVRVSRFVTWIEGVMRNN。
2. Microplasminogen. variant according to claim 1, wherein the histidine (H) of 29 sports agedoite (N)
Or/and the lysine (K) of 103 sports alanine (A).
3. Microplasminogen. variant according to claim 1, which has SEQ ID NO:4、SEQ ID NO:6、SEQ ID
NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID
NO:Sequence shown in 14.
4. the MuPlm variant that a kind of Microplasminogen. variant by described in claims 1 to 3 any one is obtained, or it is independent
The MuPlm variant corresponding with the Microplasminogen. variant described in claims 1 to 3 any one, or the fento is molten
The derivant of the derivant and MuPlm variant of proenzyme variant.
5. a kind of Microplasminogen. variant or MuPlm variant protein sequences comprising described in Claims 1-4 any one
Plasminogen or fibrinolysin, or their derivant.
6. different truncate shapes of the Microplasminogen. variant or MuPlm variant according to Claims 1-4 any one
The Microplasminogen. or MuPlm of formula, or the derivant of the variant.
7. a kind of pharmaceutical composition, containing the Microplasminogen. variant described in any one of claim 1-6, MuPlm variant,
Plasminogen variant, the derivant of fibrinolytic enzyme variants or these variants and pharmaceutically acceptable diluent, carrier or auxiliary agent.
8. pharmaceutical composition according to claim 7, which is lyophilized formulations or Injectable solution.
9. a kind of DNA sequence of Microplasminogen. variant of the coding as described in claim 1 to 6 any one.
10. a kind of expression vector containing DNA sequence as claimed in claim 9.
A kind of 11. host cells of expression vector as claimed in claim 10.
12. host cells according to claim 11, preferably pichia pastoris phaff or saccharomyces cerevisiae, or it is selected from antibacterial
Or other yeast, or zooblast etc..
The pharmaceutical composition described in MuPlm variant or claim 7 or 8 described in 13. claim 4 to 6, for preparing
Induction promotes to be dissolved by the pathologic fibrin deposition for trying internal, or for reducing recycled fibre proteinogen or using
In the enzymatic debridement of the damaged tissues of experimenter or for reducing in controlling object eye in the medicine of extraretinal neovascularization
Purposes.
The pharmaceutical composition described in MuPlm variant or claim 7 or 8 described in 14. claim 4 to 6, for preparing
Purposes in the medicine for the treatment of eye disorders, wherein described eye disorders are selected from following disease：Detachment of retina, retina break
Split, vitreous hemorrhage, diabetic vitreous hemorrhage, proliferating diabetic regard film disease, non-proliferative diabetic regard film disease,
The macular edema relevant with aging, macula lutea broken hole, the traction of vitreous body macula lutea, macular pucker, macula lutea exudate, cryptomere macula lutea water
Swollen, fibrin deposition, the retinal vein occlusion, retinal artery occlusion, subretinal hemorrhage, amblyopia, endophthalmitis, premature infant
The combination of retinopathy, glaucoma, retinitis pigmentosa and any of the above.
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| CN113355309A (en) * | 2021-08-10 | 2021-09-07 | 迈威(上海)生物科技股份有限公司 | Process for preparing recombined truncated human fibrinolysin |
| CN114480353A (en) * | 2022-01-17 | 2022-05-13 | 景泽生物医药(合肥)有限公司 | Method for preparing recombinant human akkerplasmin |
| WO2023185188A1 (en) * | 2021-03-31 | 2023-10-05 | Shenzhen Bay Laboratory | A short in vivo half-life and in vivo unstable recombinant microplasmin, pharmaceutical composition comprising thereof and method of treating thromboembolism related diseases including administration thereof |
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Cited By (5)
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| WO2023185188A1 (en) * | 2021-03-31 | 2023-10-05 | Shenzhen Bay Laboratory | A short in vivo half-life and in vivo unstable recombinant microplasmin, pharmaceutical composition comprising thereof and method of treating thromboembolism related diseases including administration thereof |
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| CN113355309A (en) * | 2021-08-10 | 2021-09-07 | 迈威(上海)生物科技股份有限公司 | Process for preparing recombined truncated human fibrinolysin |
| CN113355309B (en) * | 2021-08-10 | 2021-11-05 | 迈威(上海)生物科技股份有限公司 | Process for preparing recombined truncated human fibrinolysin |
| CN114480353A (en) * | 2022-01-17 | 2022-05-13 | 景泽生物医药(合肥)有限公司 | Method for preparing recombinant human akkerplasmin |
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|---|---|
| CN106609266B (en) | 2020-09-11 |
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